Experimental Study Of Parathyroid Hormone-Related Peptide-1/Calcium-Phosphorus Composite Scaffold For Repairing Osteoporotic Bone Defect

Part Ⅰ Preparation and characterization of functionalized bioactive CaP scaffoldsObjective Synthesis of bioactive scaffolds using calcined natural calf cancellous bone ceramics(also called True bone ceramics,TBC or calcium phosphate,CaP)and parathyroid hormone hormone-related peptide-1(PTHrP-1)as matrix materials,namely calcium phosphate matrix scaffolds and calcium phosphate matrix scaffold containing PTHrP-1(PTHrP-1@CaP).Self-assembled polydopamine(PDA)nanocoatings were modified onto the surface of CaP scaffolds to obtain PDA-modified calcium phosphate scaffolds(PDA@CaP).Subsequently,the polydopamine hybridized nanocoatings containing PTHrP-1 were modified onto the surface of CaP scaffolds to obtain functionalized calcium phosphate scaffolds(PTHrP-1@PDA@CaP,abbreviated as PP@CaP).Characterization of their relevant physicochemical properties was tested.Methods PTH(1-34)or PTHrP-1 dry powder was dissolved in 1X PBS and the CaP scaffold was rotated overnight at low temperature in the peptide-containing solution to obtain the maximum adsorption amount.The small molecule active peptide loaded scaffold,i.e.PTHrP-1@CaP scaffold,was subsequently obtained by vacuum freeze drying method.The PDA@CaP scaffold was obtained by mussel bio-inspired modification of the PDA nanocoatings on the CaP scaffold surface by self-assembly.Subsequently,CaP was immersed into the self-assembled PDA solution containing PTHrP to obtain PTHrP-1@PDA@CaP scaffolds by covalent binding and non-covalent binding functionalization.The macro,micro and nano morphologies of the scaffold materials were observed by camera,optical microscope,scanning electron microscope,Micro-CT and transmision electron microscope,and the pore size and porosity of the scaffold materials were characterized.The physicochemical properties of the functionalized CaP scaffolds were characterized by X-ray photoelectron spectroscopy,X-ray diffraction,Fourier infrared spectroscopy,and universal testing machine.Results All three constructed functionalized CaP scaffolds exhibited the expected surface morphology and microstructure.In addition,the functionalized PTHrP-1@PDA@CaP not only retained the pore structure and mechanical properties of natural porous interoperability and pro-bone formation,but also presented certain micron/nanoscale surface mineralized layers.The structure and micron/nanoscale morphology of these unique pores conferred good bioactivity to PTHrP-1@PDA@CaP and further confirmed the successful surface modification.Elemental mapping showed that the elements in PTHrP-1@PDA@CaP were uniformly distributed on the surface of the scaffold material and the Ca/P ratio was1.64,which was lower than the Ca/P ratio of natural bone tissue of 1.67,which was favorable for new bone induction and mineralization.Chemical characterization tests revealed that the PTHrP-1@PDA@CaP scaffold mainly consisted of hydroxyapatite(HA)crystalline phase and amorphous phosphate,and the components could interact with each other through non-covalent bonds such as electrostatic adsorption and hydrogen bonding.Mechanical performance tests showed that the functionalized PTHrP-1@PDA@CaP hybrid nano-coating retained the mechanical strength of the scaffold material,maintained the integrity and stability of the scaffold,provided longer-term mechanical support and structural stability for the bone defect area in vivo,and provided a stable microenvironment for further vascular growth into and repair of the defect area.The results of in vitro bioactive factor slow release experiments showed that the functionalized PTHrP-1@PDA@CaP could maintain the slow release of PTHrP for up to 28 days,achieving a relatively long period of slow release of PTHrP and playing an important role in the subsequent biological efficacy.Conclusion The functionalized PTHrP-1@PDA@CaP scaffold has effective piggybacking of bioactive factors to achieve effective action of PTHrP in the local area,which is different from the intermittent systemic administration mode of teriparatide(PTH1-34)used in the clinic.The modified composite scaffold retains good mechanical strength,which facilitates adequate mechanical support and bone regeneration repair in the defect area.These excellent properties provide a good basis for subsequent in vitro cellular assays and in vivo repair of osteoporotic bone defects.Part Ⅱ In vitro multifunctional bioactivity of functionalized calcium phosphate scaffoldsPurpose To study the effects of functionalized CaP scaffolds on cell biosafety and biological effects from in vitro.Method Primary BMSCs,BMMs,HUVECs,and Raw 264.7 were first obtained.The extracts of different process scaffold materials were prepared according to international standards,and primary BMSCs and co-cultured with the extracts.CCK-8assay was performed after a period of co-culture to verify the effect of scaffold extracts on cell proliferation,i.e.,the indirect contact method.Cells were subsequently co-cultured by direct inoculation onto the surface of functionalized calcium phosphate scaffolds,i.e.,the direct contact method.Cell adhesion,proliferation and migration status on the scaffold were observed by live-dead cell staining,cytoskeleton staining and scanning electron microscopy.The osteogenic,angiogenic,osteoclast-inhibiting,and inflammation-inhibiting properties of the cells were continued to be examined by indirect/direct contact method.Specific experiments include cell migration,ALP activity,alizarin red staining,Von Kossa staining,Trap staining,tube-forming assays,and related gene and protein expression assays.Results The prepared functionalized PTHrP-1@PDA@CaP scaffold and the infiltrate had excellent biocompatibility and significantly promoted the in vitro adhesion,proliferation,migration and extension of BMSCs and HUVECs.Assessment of cell biological behavior by indirect contact and direct contact methods revealed that PTHrP-1@PDA@CaP scaffolds could synergistically promote proliferation migration,alkaline phosphatase activity,osteogenic differentiation,and extracellular matrix mineralization of BMSCs through natural porous interoperable structures and bioactive factors,and effectively increase the expression levels of related genes and proteins;and enhance HUVECs migration,vascular ring formation and the expression of related genes and proteins.In addition to effectively promoting osteogeneiss and angiogenes,pthathe composite functionalized calcium phosphate scaffold also inhibited the osteoclastic differentiation of BMMs and the expresinon of related genes and proteins,and inhibited the polarization of pro-inflammatory M1 macrophages and the expression of related genes.Conclusion The functionalized PTHrP-1@PDA@CaP scaffold can effectively enhance the biocompatibility of BMSCs and HUVECs in vitro in a structure-and composition-dependent manner,promote osteogenic differentiation and angiogenic potential of cells in vitro,inhibit osteolytic activity and pro-inflammatory M1 macrophage polarization,and lay a scientific foundation for further evaluation of this functionalized calcium phosphate scaffold material in osteoporotic bone defect repair in vivo.Part Ⅲ In vivo bone regeneration in osteoporotic bone defects by functionalized CaP scaffoldsPurpose To further explore the feasibility of functionalized CaP scaffolds in the repair of osteoporotic bone defects in vivo and to evaluate their in vivo pro-osteogenic and osseointegrative abilities.Method Twelve 12-week-old SD female rats were first taken for the construction and validation of an osteoporotic cranial bone defect model.The rats were randomly divided into control and ovary removal(VOX)groups.After 12 weeks of ovary removal,Micro-CT scanning and 3D reconstruction,pathological staining of tissue sections,and immunohistochemical staining were performed on femur specimens to verify the successful construction of the osteoporosis model.After that,30 female rats were selected,and both ovaries of each rat were removed in the first step,and the second step was performed 12 weeks later.In the second step,a critical defect model with a bilateral diameter of 5 mm in the rat skull was constructed,and the rats were randomly divided into 5 groups according to the type of implant material in each group: CaP group,PTH@CaP group,iPTH+PDA@CaP group,PTHrP-1@CaP group,and PTHrP-1@PDA@CaP group.The cranial specimens were removed at week 12 two after stent implantation.The effects of functionalized calcium phosphate scaffolds on new bone production and bone repair in vivo were investigated by gross morphological observation,Micro-CT scanning and three-dimensional reconstruction,pathological staining of tissue sections,immunohistochemical staining and immunofluorescence staining.Result Femoral specimens were taken from rats 12 weeks after ovariectomy for Micro-CT scanning.The results showed that the VOX group had fewer trabeculae,disturbed trabecular structure,thinning of the bone cortex,and only a small amount of new bone production.The results of H&E and Goldner staining further confirmed that the trabeculae were sparse and disorganized,with more adipose tissue filling the bone marrow cavity.Immunohistochemical results further confirmed reduced osteogenic activity and strong osteolytic activity in the VOX group,with a polarization of the immune microenvironment towards pro-inflammatory M1 macrophages.All implanted scaffold materials had good biocompatibility in vivo and did not adversely affect the major organs of the rats.Micro-CT scans and 3D reconstruction analyiss showed that the calcium phosphate-based scaffold had a strong ability to guide the directional growth of bone tissue and accelerate the healing of bone defects.At 12 weeks postoperatively,new bone in the PTHrP-1@PDA@CaP group covered almost the entire defect area and crawled along the natural void resultant replacement,demonstrating the guiding effect of calcium phosphate-based scaffolds.The results of H&E,Masson and Goldner staining showed that compared with the control group,both scaffolds,significantly promoted the bone defect area within the regeneration of vascularized bone tissue,and also improved the mineralization and maturation of new bone.Among them,the PTHrP-1@PDA@CaP scaffold had the most significant promotion effect,which was also confirmed by the immunohistochemical results,i.e.,the osteogenic and angiogenic-related protein indices were significantly increased in this scaffold group.Conclusion We successfully constructed an osteoporotic bone defect model,and the functionalized PTHrP-1@PDA@CaP scaffold has the effect of promoting the growth of new bone into and the formation of blood vessels in the area of cranial bone defects in rats.By preparing structurally and compositionally optimized PTHrP-1@PDA@CaP scaffolds,a positive contribution to vascularized bone regeneration could be achieved.